Isolation and expression of murine carbonic anhydrase IV.
A 1193-bp cDNA containing the complete murine carbonic anhydrase IV coding sequence was isolated from a Balb/c kidney cDNA library. The entire coding sequence plus shorter segments was used in an Escherichia coli T7 expression vector system to produce four forms of murine CA IV, including (1) a protein representing the full-length coding sequence, (2) an amino-truncated protein lacking the 18 N-terminal amino acid plasma membrane targeting sequence, (3) a protein which lacked the plasma membrane targeting sequence and 26 C-terminal amino acids, and (4) a protein which lacked both 36 N-terminal residues (the plasma membrane targeting sequence plus 18 additional amino acids which included the first two cysteines) and 26 C-terminal residues. All four proteins were expressed as catalytically inactive inclusion bodies. After rapid dilution of washed, guanidine hydrochloride-denatured inclusion bodies into a glutathione-, l-arginine-containing renaturation buffer, an active carbonic anhydrase IV at yields of 3-4 mg/liter was easily purified from cultures expressing the form lacking the N-terminal targeting sequence and 26 C-terminal residues. The longest and shortest forms of carbonic anhydrase IV failed to refold into active enzyme under these conditions. The activity of purified recombinant carbonic anhydrase IV was highly resistant to sodium dodecyl sulfate, as is the native enzyme. This resistance presumably results from intramolecular disulfide bonds maintaining a functional active site configuration even in the presence of denaturing agents.
Hurt
J D
JD
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, Florida 32610, USA.
Tu
C
C
Laipis
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D017510:Protein Folding; D051381:Rats; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D002256:Carbonic Anhydrases